Strain selection of pneumococcal surface proteins

ABSTRACT

The present invention relates to vaccine composition(s) comprising at least two PspAs from strains selected from at least one family, the family being defined by PspAs from strains belonging to the family having greater than or equal to 50% homology in aligned sequences of a C-terminal region of an alpha helical region of PspA. Additionally, the families are further comprised of clades, wherein PspAs from strains which belong to a clade exhibit at least 75% sequence homology in aligned sequences of the C-terminal region of the alpha helix of PspA. Vaccine compositions of the present invention preferably comprise a minimum of 4 and a maximum of 6 strains representing a single clade each, and the at least two PspAs are optionally serologically or broadly cross-reactive.

RELATED APPLICATIONS

This application is a continuation-in-part (“CIP”) of U.S. Ser. No.08/710,749, filed Sep. 20, 1996, now U.S. Pat. No. 5,955,089, and a U.S.National Application of PCT application Ser. No. PCT/US97/16761, filedSep. 22, 1997.

DESCRIPTION OF DEPOSITED BIOLOGICAL MATERIALS

The Streptococcus pneumoniae strain designated Rx1 has been depositedpursuant to, and in satisfaction of, the requirements of the BudapestTreaty on the International Recognition of The Deposit of Microorganismsfor The Purposes of Patent Procedure, with the American Type CultureCollection (ATCC), now at 10801 University Boulevard, Manassas, Va.20110-2209, under ATCC Accession No. 55834, on Oct. 3, 1996.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is an important cause of otitis media,meningitis, bacteremia and pneumonia, and a leading cause of fatalinfections in the elderly and persons with underlying medical conditionssuch as pulmonary disease, liver disease, alcoholism, sickle cellanemia, cerebrospinal fluid leaks, acquired immune deficiency syndrome(AIDS), and patients undergoing immunosuppressive therapy. It is also aleading cause of morbidity in young children. Pneumococcal infectionscause approximately 40,000 deaths in the U.S. each year. The most severepneumococcal infections involve invasive meningitis and bacteremiainfections, of which there are 3,000 and 50,000 cases annually,respectively.

Despite the use of antibiotics and vaccines, the prevalence ofpneumococcal infections has declined little over the last twenty-fiveyears; the case-fatality rate for bacteremia is reported to be 15-20% inthe general population, 30-40% in the elderly, and 36% in inner-cityAfrican Americans. Less severe forms of pneumococcal disease arepneumonia, of which there are 500,000 cases annually in the U.S., andotitis media in children, of which there are an estimated 7,000,000 ofsuch cases each year are caused by pneumococcus. Strains ofdrug-resistant S. pneumoniae are becoming ever more common in the U.S.and worldwide. In some areas, as many as 30% of pneumococcal isolatesare resistant to penicillin. The increase in antimicrobial resistantpneumococcus further emphasizes the need for preventing pneumococcalinfections.

Pneumococcus asymptomatically colonizes the upper respiratory tract ofnormal individuals; disease often results from the spread of organismsfrom the nasopharynx to other tissues during opportunistic events. Theincidence of carriage in humans varies with age and circumstances.Carrier rates in children are typically higher than those of adults.Studies have demonstrated that 38 to 60% of preschool children, 29 to35% of grammar school children and 9 to 25% of junior high schoolchildren are carriers of pneumococcus. Among adults, the rate ofcarriage drops to 6% for those without children at home, and to 18 to29% for those with children at home. It is not surprising that thehigher rate of carriage in children than in adults parallels theincidence of pneumococcal disease in these populations.

An attractive goal for streptococcal vaccination is to reduce carriagein the vaccinated populations and subsequently reduce the incidence ofpneumococcal disease. There is speculation that a reduction inpneumococcal carriage rates by vaccination could reduce the incidence ofthe disease in non-vaccinated individuals as well as vaccinatedindividuals. This “herd immunity” induced by vaccination against upperrespiratory bacterial pathogens has been observed using the Haemophilusinfluenzae type b conjugate vaccines (Takala, A. K., et al., J. Infect.Dis. 1991; 164: 982-986; Takala, A. K., et al., Pediatr. Infect. Dis.J., 1993; 12: 593-599; Ward, J., et al., Vaccines, S. A. Plotkin and E.A. Mortimer, eds., 1994, pp. 337-386; Murphy, T. V., et al., J.Pediatr., 1993; 122; 517-523; and Mohle-Boetani, J. C., et al., Pediatr.Infect. Dis. J., 1993; 12: 589-593).

It is generally accepted that immunity to Streptococcus pneumoniae canbe mediated by specific antibodies against the polysaccharide capsule ofthe pneumococcus. However, neonates and young children fail to makeadequate immune response against most capsular polysaccharide antigensand can have repeated infections involving the same capsular serotype.One approach to immunizing infants against a number of encapsulatedbacteria is to conjugate the capsular polysaccharide antigens to proteinto make them immunogenic. This approach has been successful, forexample, with Haemophilus influenzae b (see U.S. Pat. No. 4,496,538 toGordon and U.S. Pat. No. 4,673,574 to Anderson).

However, there are over ninety known capsular serotypes of S.pneumoniae, of which twenty-three account for about 95% of the disease.For a pneumococcal polysaccharide-protein conjugate to be successful,the capsular types responsible for most pneumococcal infections wouldhave to be made adequately immunogenic. This approach may be difficult,because the twenty-three polysaccharides included in thepresently-available vaccine are not all adequately immunogenic, even inadults.

Protection mediated by anti-capsular polysaccharide antibody responsesare restricted to the polysaccharide type. Different polysaccharidetypes differentially facilitate virulence in humans and other species.Pneumococcal vaccines have been developed by combining 23 differentcapsular polysaccharides that are the prevalent types of humanpneumococcal disease. These 23 polsaccharide types have been used in alicensed pneumococcal vaccine since 1983 (D. S. Fedson and M. Musher,Vaccines, S. A. Plotkin and J. E. A. Montimer, eds., 1994, pp. 517-564).The licensed 23-valent polysaccharide vaccine has a reported efficacy ofapproximately 60% in preventing bacterermia caused by vaccine typepneumococci in healthy adults.

However, the efficacy of the vaccine has been controversial, and attimes, the justification for the recommended use of the vaccinequestioned. It has been speculated that the efficacy of this vaccine isnegatively affected by having to combine 23 different antigens. Having alarge number of antigens combined in a single formulation may negativelyaffect the antibody responses to individual types within this mixturebecause of antigenic competition. The efficacy is also affected by thefact that the 23 serotypes encompass all serological types associatedwith human infections and carriage.

An alternative approach for protecting children, and also the elderly,from pneumococcal infection would be to identify protein antigens thatcould elicit protective immune responses. Such proteins may serve as avaccine by themselves, may be used in conjunction with successfulpolysaccharide-protein conjugates, or as carriers for polysaccharides.

McDaniel et al. (I), J. Exp. Med. 160:386-397, 1984, relates to theproduction of monoclonal antibodies that recognize cell surfacepolypeptide(s) on S. pneumoniae and protection of mice from infectionwith certain strains of encapsulated pneumococci by such antibodies.

This surface protein antigen has been termed “pneumococcal surfaceprotein A”, or “PspA” for short.

McDaniel et al. (II), Microbial Pathogenesis 1:519-531, 1986, relates tostudies on the characterization of the PspA. Considerable diversity inthe PspA molecule in different strains was found, as were differences inthe epitopes recognized by different antibodies.

McDaniel et al. (III), J. Exp. Med. 165:381-394, 1987, relates toimmunization of X-linked immunodeficient (XID) mice withnon-encapsulated pneumococci expressing PspA protects mice fromsubsequent fatal infection with pneumococci, but immunization withisogenic pneumococci which do not express PspA does not conferprotection.

McDaniel et al. (IV), Infect. Irnmun., 59:222-228, 1991, relates toimmunization of mice with a recombinant full length fragment of PspAthat is able to elicit protection against pneumococcal strains ofcapsular types 6A and 3.

Crain et al, Infect.Immun., 56:3293-3299, 1990, relates to a rabbitantiserum that detects PspA in 100% (n=95) of clinical and laboratoryisolates of strains of S. pneumoniae. When reacted with seven monoclonalantibodies to PspA, fifty-seven S. pneumoniae isolates exhibitedthirty-one different patterns of reactivity.

U.S. Pat. No. 5,476,929, relates to vaccines comprising PspA andfragments thereof, methods for expressing DNA encoding PspA andfragments thereof, DNA encoding PspA and fragments thereof, the aminoacid sequences of PspA and fragments thereof, compositions containingPspA and fragments thereof and methods of using such compositions.

PspA has been identified as a virulence factor and protective antigen.PspA is a cell surface molecule that is found on all clinical isolates,and the expression of PspA is required for the full virulence ofpneumococci in mouse models (McDaniel et al., (III), J. Exp. Med. 165:381-394, 1987). The biological function of PspA has not been welldefined. although a preliminary report suggests that it may inhibitcomplement activation (Alonso DeVelasco, E., et al., MicrobiologicalRev. 1995; 59: 591-603).

The PspA protein type is independent of capsular type. It would seemthat genetic mutation or exchange in the environment has allowed for thedevelopment of a large pool of strains which are highly diverse withrespect to capsule, PspA, and possibly other molecules with variablestructures. Variability of PspA's from different strains also is evidentin their molecular weights, which range from 67 to 99 kD. The observeddifferences are stably inherited and are not the result of proteindegradation.

Immunization with PspA in a lysate of a recombinant lgt11 clone,elicited protection against challenge with several S. pneumoniae strainsrepresenting different is capsular and PspA types, as in McDaniel et al.(IV), Infect. Immun. 59:222-228, 1991.

Although clones expressing PspA were constructed according to thatpaper, the product was insoluble and isolation from cell fragmentsfollowing lysis was not possible.

Analysis of the nucleotide and amino acid sequences of the PspA moleculereveals three major regions. The first 288 amino acids at the aminoterminal end of the protein are predicted to have a strong alpha helicalstructure. The adjacent region of 90 amino acids (289 to 369 of Rx1PspA) has a high density of proline residues; based on similar regionsin other prokaryotic proteins, this region is believed to traverse thebacterial cell wall. The remaining 196 amino acids at thecarboxyl-terminal end of the molecule (370 to 588 of Rx1 PspA) have arepeated amino acid sequence that has been demonstrated to bind tophosphocholine and lipoteichoic acids. Based on this structure, the PspAmolecule is thought to associate with the inner membrane andlipoteichoic acids via the repeated region in the middle of thecarboxyl-terminal end of the protein. The proline region in the middleof the protein is thought to traverse the cell wall, placing the alphahelical region on the outer surface of the S. pneumoniae cells. Thismodel is consistent with the demonstration that the alpha helicalregion, which extends from the surface of the cell, contains theprotective epitopes (Yother, J. et al., J. Bacteriol. 1992; 174:601-609; Yother, J. et al., J. Bacteriol. 1994; 176: 2976-2985;McDaniel, L. S. et al., Microbial Pathog. 1994; 17: 323-337; and Ralph,B. A., et al., Ann. N. Y. Acad. Sci. 1994; 730: 361-363).

Serological analysis of PspA using a panel of seven monoclonalantibodies, indicated that, like capsular polysaccharides, the PspAmolecules are highly diverse among pneumococcal strains. Based on theseanalyses, over 30 PspA protein serotypes were defined, and individualstrains were assigned into groups, i.e., families (or serotypes) using aclassification system based upon reactivity with the panel of monoclonalantibodies. Moreover, SDS-PAGE analysis indicated that, within a PspAserotype, further heterogeneity existed on the basis of the molecularsize. This diversification further supports the assertion that PspA is aprotective antigen in natural infections; the protective nature ofanti-PspA responses has presumably applied selective pressure onpneumococcus to diversify this molecule. However, this diversificationof the PspA molecule complicates the development of a PspA vaccine, andleads to the possibility that a PspA vaccine would have to contain manyPspA strains, possibly making the vaccine impractical.

Briles et al., PCT 92/000857, used a pspA-specific probe to identifyrelated proteins among different strains of S. pneumonia. One suchPspA-like polypeptide has designated PspC et al., Abstracts of the 97thAnnual Meeting of the American Societies for Microbiology, May 1997. Thegene encoding PspC hybridizes to a full-length pspA probe, demonstratingthe close relatedness of the PspA and PspC proteins at the molecularlevel. Comparison of consensus sequences for the PspA clades with knowpspC genes indicates that some of the PspC proteins can be classifiedwithin the defined PspA clades. In fact, sequence analysis of pspC genesfrom distinct isolates of S. pneumoniae reveals a greater than 85%homology at the amino acid level between the products of these pspCgenes and those of pspA genes from representatives of Clade 2.Furthermore, PspC contains the same three major regions describedhereinabove for PspA, namely an alpha helical N-terminal domain, aproline-rich region, and a choline binding C-terminal domain. Also,polyclonal antibodies raised against PspC cross-react with PspAproteins. Thus, for the purposes of the present invention, the term“PspA”, as it appears in the specifications and in the claims appendedthereto, includes full-length and truncated forms ofnaturally-occurring, synthetic, semi-synthetic or recombinant forms ofPspA of PspC.

In addition to the published literature specifically referred to above,the inventors, in conjunction with co-workers, have published furtherdetails concerning PspA's, as follows:

1. Abstracts of 89th Annual Meeting of the American Society forMicrobiology, p. 125, item D-257, May 1989;

2. Abstracts of 90th Annual Meeting of the American Society forMicrobiology, p. 98, item D-106, May 1990;

3. Abstracts of 3rd International ASM Conference on StreptococcalGenetics, p. 11, item 12, June 1990;

4. Talkigton et al, Infect. Immun. 59:1285-1289, 1991;

5. Yother et al (I), J. Bacteriol. 174:601-609, 1992; and

6. Yother et al (II), J. Bacteriol. 174:610-618, 1992.

7. McDaniel et al (V), Microbiol. Pathogenesis, 13:261-268, 1994.

Alternative vaccination strategies are desirable as such providealternative routes to administration or alternative routes to generationof immune responses. It would be advantageous to provide animmunological composition or vaccination regimen which elicitsprotection against various diverse pneumococcal strains, without havingto combine a large number of possibly competitive antigens within thesame formulation.

The prior art fails to provide broadly efficacious pneumococcalvaccines. Suprisingly, the present inventions technique of clade andfamily groups within the Pneumococci solves this deficiency of prior artapproaches by allowing a rational selection of representative PspAs fromthe various families of clades to produce broadly efficaciousPneumococcal vaccines, reagents and methods.

The present invention provides a vaccine composition comprising at leasttwo PspAs from strains selected from at least two families. A family isdefined by PspAs from strains having greater than or equal to 50%homology in aligned sequences of a C-terminal region of an alpha helixof PspA.

The invention provides vaccine compositions, wherein the familiesfurther comprise one or more clades. Clades are defined by PspAs havingat least 75% homology with other PspAs from a strain within the clade inthe aligned sequences of the C-terminal region of the alpha helix ofPspA.

Additionally, the present invention provides vaccine compositionswherein the C-terminal region of PspA contains epitope(s) of interest.

The present invention further provides vaccine compositions wherein acentral domain comprising the C-terminal 100 amino acids of thealpha-helical region (192 to 290 of Rx1 PspA) contains epitope(s)capable of eliciting a protective response.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a Pustell DNA matrix analysis of homology between the PspAgenes of Rx1 and EF10197 strains;

FIG. 2 shows a Pustell protein matrix analysis of homology between thePspA proteins of Rx1 and EF10197 strains;

FIG. 3 shows the alignment of PspA protein amino acid sequences from theBG6692 strain (SEQ ID NO: 2), the BG8838 strain (SEQ ID NO: 3), theBG9739 strain (SEQ ID NO: 4), the DBL6A strain (SEQ ID NO: 5), theL81905 strain (SEQ ID NO: 6), the BG8743 strain (SEQ ID NO: 7), the DBL1strain (SEQ ID NO: 8), and the AC94 strain (SEQ ID NO: 9) to the PspAclade 1 consensus sequence (SEQ ID NO: 1). FIG. 3 corresponds to thedata presented in Table 3.

FIG. 4 shows the alignment of PspA protein amino acid sequences from theEF10197 strain (SEQ ID NO: 11), the Rx1 strain (SEQ ID NO: 12), the WU2strain (SEQ ID NO: 13), the DBL5 strain (SEQ ID NO: 14), the EF6796strain (SEQ ID NO: 15), the 0922134 strain (SEQ ID NO: 16), and theBG9163 strain (SEQ ID NO: 17) to the PspA clade 2 consensus sequence(SEQ ID NO: 10). FIG. 4 corresponds to the data presented in Table 4.

FIG. 5 shows the alignment of PspA protein amino acid sequences from theAC122 strain (SEQ ID NO: 19), the EF3296 strain (SEQ ID NO: 20), and theBG8090 strain (SEQ ID NO: 21) to the PspA clade 3 consensus sequence(SEQ ID NO: 18). FIG. 5 corresponds to the data presented in Table 5.

FIG. 6 shows the alignment of PspA protein amino acid sequences from theBG11703 strain (SEQ ID NO: 23), the BG7817 strain (SEQ ID NO: 24), theEF5668 strain (SEQ ID NO: 25), and the BG7561 strain (SEQ ID NO: 26) tothe PspA clade 4 consensus sequence (SEQ ID NO: 22). FIG. 6 correspondsto the data presented in Table 6.

FIG. 7 shows the amino acid sequence of PspA from strain ATCC6303 (SEQID NO: 27), a representative strain of clade 5.

FIG. 8 shows the amino acid sequence of PspA from strain BG6380 (SEQ IDNO: 28), a representative strain of clade 6.

FIG. 9 shows the competitive inhibition of rabbit polyclonal anti-Rx1 byPA314, recombinant Rx1 containing amino acids 96 to 314;

FIG. 10 shows the inhibition of polyclonal rabbit anti-Rx1 antibodies byPARx1 and PAEF5668 antigens; and

FIG. 11 shows the inhibition of polyclonal rabbit anti-Rx1 antibodies byPARx1 and PABG6380 antigens.

DETAILED DESCRIPTION

It has now been surprisingly found that, despite the assertions of theprior art regarding the apparent diversity of PspA from strains, theprimary sequence of the alpha helix of PspA has two regions of relativeconservation and a region of extensive diversity between PspAs fromstrains. The two regions of relative conservation are comprised of thefirst, N-terminal, 60 amino acids of the alpha helix, and the last,C-terminal, about 100 amino acids of the alpha helix, as shown in FIGS.1 and 2, wherein the C-terminal end of the alpha helix containscross-reactive and protective epitopes that are critical to thedevelopment of a broadly efficacious PspA vaccine. It has been foundthat any conservation in the first, N-terminal, 60 amino acids of thealpha helix is of little onsequence in the cross-reactivity of thestrain, and hence, is irrelevant to the evelopment of a PspA vaccine.

A comparison of the amino acid sequences in the C-terminal region of thealpha helix of PspAs from 24 strains of S. pneumoniae has revealed thatthe PspA strains can be grouped into 6 clades with greater than 75%homology, and these clades can be grouped into 4 families with greaterthan 50% homology.

Accordingly, the present invention provides a method of strain selectionof PspA, based upon the sequence homology of PspAs in the C-terminalregion of the alpha helix.

A clade is defined herein as comprising PspAs which exhibit greater than75% sequence homology in aligned sequences of the C-terminal region ofthe alpha helix, and a family is defined herein as those clades whichexhibit greater than or equal to 50% homology between member PspAsequences in aligned sequences of the C-terminal region of the alphahelix.

Further, it has been found that in addition to sequence homology,members of a clade exhibit cross-reactivity and cross-protection amongone another, which may suggest a causal relationship between sequencehomology and cross-reactivity. PspAs of strains within the same PspAclade exhibit reciprocal cross-protection from immunization andchallenge experiments. It has not been heretofore recognized in theprior art that there may be such a causal relationship; in fact,families of PspA strains were defined solely on the basis of serologicalcross-reactivity and, based upon the prior art definition of families ofPspA strains, it was believed that the extreme diversity of the PspAmolecule would result in a futile attempt at strain selection. Moreover,the PspA typing system (Crain, et al., Infect. Immun. 59: 222-228, 1990)failed to provide relevant groupings of strains, and suggested anenormous diversity.

Hence, the present invention, in contrast to the teachings of the priorart, enables the selection of PspAs from strains in accordance withsequence homology and cross-reactivity, which facilitates thedevelopment of vaccine compositions comprising multiple PspAs.

The present invention contemplates vaccine compositions comprising twoor more, preferably no more than 10, and more preferably a minimum of 4and a maximum of 6 strains of PspA representing a single clade each, anda pharmaceutically acceptable carrier or diluent. In a preferredembodiment, Rx1, a member of clade 2, is the preferred strain of clade 2and/or family 1 which is optionally included in the vaccine compositionof the present invention.

The aforementioned definition of a family structure to the C-terminalend of the alpha helix region enables the development of a broadlyefficacious pneumococcal vaccine composition with a limited number ofstrains. Combining strains that represent some or all of the familiesfor this cross-reactive and protective region should provide broadprotection against pneumococcal disease. Not all clades may need to berepresented because of cross-reactions between some clades withinfamilies or because of the epidemiology of these strains or clades inthe population to be vaccinated. However, it is well within the scope ofknowledge of the skilled artisan to determine those strains which shouldbe included within a vaccine composition, without the burden of undueexperimentation. Additionally, the selected PspAs and PspA-likepolypeptides of the present invention further contain epitope(s) ofinterest which can elicit an immune response. An epitope of interest isthat portion of an antigen or immunogen of interest which is capable ofinteracting with an antibody or T cell.

The present invention provides an immunogenic, immunological or vaccinecomposition containing pneumococcal strain(s) having an epitope ofinterest, and a pharmaceutically acceptable carrier or diluent. Animmunological composition containing the PspAs having an epitope ofinterest, elicits an immunological response—local or systemic. Theresponse can, but need not be, protective. An immunogenic compositioncontaining the PspAs having an epitope of interest, likewise elicits alocal or systemic immunological response which can, but need not be,protective. A vaccine composition elicits a local or systemic protectiveresponse. Accordingly, the terms “immunological composition” and“immunogenic composition” include a “vaccine composition” (as the twoformer terms can be protective compositions).

The invention therefore also provides a method of inducing animmunological response in a host mammal comprising administering to thehost an immunogenic, immunological or vaccine composition comprisingPspAs having an epitope of interest, and a pharmaceutically acceptablecarrier or diluent. As to epitopes of interest, one skilled in the artcan often identify epitopes or immunodominant regions of a peptide orpolypeptide, and ergo, the coding DNA therefor from the knowledge of theamino acid and corresponding DNA sequences of the peptide orpolypeptide, as well as from the nature of particular amino acids (e.g.,size, charge, etc.) and the codon dictionary, without undueexperimentation.

A general method for determining which portions of a protein to use inan immunological composition focuses on the size and sequence of theantigen of interest. “In general, large proteins, because they have morepotential determinants are better antigens than small ones. The moreforeign an antigen, that is the less similar to self configurationswhich induce tolerance, the more effective it is in provoking an immuneresponse.” Ivan Roitt, Essential Immunology, 1988.

At a minimum, such a peptide must be at least 8 or 9 amino acids long.This is the minimum length that a peptide needs to be in order tostimulate a CD4+ T cell response (which recognizes virus infected cellsor cancerous cells). A minimum peptide length of 13 to 25 amino acids isuseful to stimulate a CD8+ T cell response (which recognizes specialantigen presenting cells which have engulfed the pathogen). See Kendrew,supra. However, as these are minimum lengths, these peptides are likelyto generate an immunological response, i.e., an antibody or T cellresponse; but, for a protective response (as from a vaccinecomposition), a longer peptide is preferred.

With respect to the sequence, the DNA sequence encoding the immunogenicpeptide preferably encodes at least regions of the peptide that generatean antibody response or a T cell response. One method to determine T andB cell epitopes involves epitope mapping. The protein of interest “isfragmented into overlapping peptides with proteolytic enzymes. Theindividual peptides are then tested for their ability to bind to anantibody elicited by the native protein or to induce T cell or B cellactivation. This approach has been particularly useful in mapping T-cellepitopes since the T cell recognizes short linear peptides complexedwith MHC molecules. The method is less effective for determining B-cellepitopes” since B cell epitopes are often not linear amino acid sequencebut rather result from the tertiary structure of the folded threedimensional protein. Janis Kuby, Immunology, (1992) pp. 79-80.

In the case of PspA, the location of the major cross-reactive region atthe C-terminal 100 amino acids of the alpha-helical region was carriedout with recombinant peptides of 100 or more amino acids in length(McDaniel et al., Micro. Pathog. 17: 323-337, 1994).

Another method for determining an epitope of interest is to choose theregions of the protein that are hydrophilic. Hydrophilic residues areoften on the surface of the protein and therefore often the regions ofthe protein which are accessible to the antibody. Janis Kuby,Immunology, (1992) P. 81.

Yet another method for determining an epitope of interest is to performan X-ray crystallographic analysis of the antigen (full length)-antibodycomplex. Janis Kuby, Immunology, (1992) p. 80.

An immune response is generated, in general, as follows: T cellsrecognize proteins only when the protein has been cleaved into smallerpeptides, which are then presented to the T cells in a complex calledthe major histocompatability complex (“MHC”) located on another cell'ssurface. There are two classes of MHC complexes-class I and class II,and each class is made up of many different alleles. Different patientshave different types of MHC complex alleles; they are said to have adifferent “HLA type”.

Class I MHC complexes are found on virtually every cell and presentpeptides from proteins produced inside the cell. Thus, Class I MHCcomplexes are useful for killing cells which have been infected byviruses or which have become cancerous and as the result of expressionof an oncogene. T cells which have a protein called CD4 on their surfacebind to the MHC class I cells and secrete lymphokines. The lymphokinesstimulate a response; cells arrive and kill the virus-infected cell.Class II MHC complexes are found only on antigen-presenting cells andare used to present peptides from circulating pathogens which have beenendocytosed by the antigen-presenting cells. T cells which have aprotein called CD8 bind to the MHC class II cells and kill the cell byexocytosis of lytic granules.

Thus, another method for identifying epitopes of interest is to identifythose regions of the protein which can generate a T cell response. Inorder to generate a T cell response, a peptide which comprises aputative epitope should be presented in the context of a MHC complex.Those skilled in the art can identify from the protein sequence of theantigen of interest potential human lymphocyte antigen (“HLA”) anchorbinding motifs. HLA anchor binding motifs are peptide sequences whichare known to be likely to bind to the MHC molecule. The peptidepreferably contains appropriate anchor motifs for binding to the MHCmolecules, and should bind with high enough affinity to generate animmune response. Other factors which can be considered are: the HLA typeof the patient (vertebrate, animal or human) expected to be immunized,the sequence of the protein, the presence of appropriate anchor motifsand the occurrence of the peptide sequence in other vital cells.

Some guidelines in determining whether a protein contains epitopes ofinterest capable of stimulating a T cell response, include: Peptidelength—the peptide should be at least 8 or 9 amino acids long to fitinto the MHC class I complex and at least 13-25 amino acids long to fitinto a class II MHC complex. This length is a minimum for the peptide tobind to the MHC complex. It is preferred for the peptides to be longerthan these lengths because cells may cut the expressed peptides. Thepeptide should contain an appropriate anchor motif which will enable itto bind to the various class I or class II molecules with high enoughspecificity to generate an immune response (See Bocchia, M. et al,Specific Binding of Leukemia Oncogene Fusion Protein Peptides to HLAClass I Molecules, Blood 85:2680-2684; Englehard, VH, Structure ofpeptides associated with class I and class II MHC molecules Ann. Rev.Immunol. 12:181 (1994)). This can be done, without undueexperimentation, by comparing the sequence of the protein of interestwith published structures of peptides associated with the MHC molecules.Protein epitopes recognized by T cell receptors are peptides generatedby enzymatic degradation of the protein molecule and are presented onthe cell surface in association with class I or class II MHC molecules.

Further, the skilled artisan can ascertain an epitope of interest bycomparing the protein sequence with sequences listed in the protein database. Regions of the protein which share little or no homology arebetter choices for being an epitope of that protein and are thereforeuseful in a vaccine or immunological composition. Regions which sharegreat homology with widely found sequences present in vital cells shouldbe avoided.

Even further, another method is simply to generate or express portionsof a protein of interest, generate monoclonal antibodies to thoseportions of the protein of interest, and then ascertain whether thoseantibodies inhibit growth in vitro of the pathogen from which theprotein was derived. The skilled artisan can use the other guidelinesset forth in this disclosure and in the art for generating or expressingportions of a protein of interest for analysis as to whether antibodiesthereto inhibit growth in vitro. For example, the skilled artisan cangenerate portions of a protein of interest by: selecting 8 to 9 or 13 to25 amino acid length portions of the protein, selecting hydrophilicregions, selecting portions shown to bind from X-ray data of the antigen(full length)-antibody complex, selecting regions which differ insequence from other proteins, selecting potential LA anchor bindingmotifs, or any combination of these methods or other methods known inthe art.

Epitopes recognized by antibodies are expressed on the surface of aprotein. To determine the regions of a protein most likely to stimulatean antibody response one skilled in the art can preferably perform anepitope map, using the general methods described above, or other mappingmethods known in the art.

As can be seen from the foregoing, without undue experimentation, fromthis disclosure and the knowledge in the art, the skilled artisan canascertain the amino acid and corresponding DNA sequence of an epitope ofinterest for obtaining a T cell, B cell and/or antibody response. Inaddition, reference is made to Defter et al., U.S. Pat. No. 5,019,384,issued May 28, 1991, and the documents it cites, incorporated herein byreference (Note especially the “Relevant Literature” section of thispatent, and column 13 of this patent which discloses that: “A largenumber of epitopes have been defined for a wide variety of organisms ofinterest. Of particular interest are those epitopes to whichneutralizing antibodies are directed. Disclosures of such epitopes arein many of the references cited in the Relevant Literature section.”)

Further, the invention demonstrates that more than one serologicallycomplementary PspA molecule can be in an antigenic, immunological orvaccine composition, so as to elicit better response, e.g., protection,for instance, against a variety of strains of pneumococci; and, theinvention provides a system of selecting PspAs for a multivalentcomposition which includes cross-protection evaluation so as to providea maximally efficacious composition. The determination of the amount ofantigen, e.g., PspA or truncated portion thereof and optional adjuvantin the inventive compositions and the preparation of those compositionscan be in accordance with standard techniques well known to thoseskilled in the pharmaceutical or veterinary arts. In particular, theamount of antigen and adjuvant in the inventive compositions and thedosages administered are determined by techniques well known to thoseskilled in the medical or veterinary arts taking into consideration suchfactors as the particular antigen, the adjuvant (if present), the age,sex, weight, species and condition of the particular animal or patient,and the route of administration. For instance, dosages of particularPspA antigens for suitable hosts in which an immunological response isdesired, can be readily ascertained by those skilled in the art fromthis disclosure, as is the amount of any adjuvant typically administeredtherewith. Thus, the skilled artisan can readily determine the amount ofantigen and optional adjuvant in compositions and to be administered inmethods of the invention. Typically, an adjuvant is commonly used as0.001 to 50 wt. % solution in phosphate buffered saline, and the antigenis present on the order of micrograms to milligrams, such as about0.0001 to about 5 wt. %, preferably about 0.0001 to about 1 wt. %, mostpreferably about 0.0001 to about 0.05 wt. % (see, e.g., Examples belowor in applications cited herein). Typically, however, the antigen ispresent in an amount on the order of micrograms to milligrams, or, about0.001 to about 20 wt. %, preferably about 0.01 to about 10 wt. %, andmost preferably about 0.05 to about 5 wt. %.

Of course, for any composition to be administered to an animal or human,including the components thereof, and for any particular method ofadministration, it is preferred to determine therefor: toxicity, such asby determining the lethal dose (LD) and LD₅₀ in a suitable animal modele.g., rodent such as mouse; and, the dosage of the composition(s),concentration of components therein and timing of administering thecomposition(s), which elicit a suitable immunological response, such asby titrations of sera and analysis thereof for antibodies or antigens,e.g., by ELISA and/or RFFIT analysis. Such determinations do not requireundue experimentation from the knowledge of the skilled artisan, thisdisclosure and the documents cited herein. And, the time for sequentialadministrations can be ascertained without undue experimentation.

Examples of compositions of the invention include liquid preparationsfor orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric,mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratorymucosa) etc., administration such as suspensions, syrups or elixirs;and, preparations for parenteral, subcutaneous, intradermal,intramuscular or intravenous administration (e.g., injectableadministration), such as sterile suspensions or emulsions. Suchcompositions may be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose or thelike. The compositions can also be lyophilized. The compositions cancontain auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation.

Compositions of the invention are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions which may be buffered to a selected pH. Ifdigestive tract absorption is preferred, compositions of the inventioncan be in the “solid” form of pills, tablets, capsules, caplets and thelike, including “solid” preparations which are time-released or whichhave a liquid filling, e.g., gelatin covered liquid, whereby the gelatinis dissolved in the stomach for delivery to the gut. If nasal orrespiratory (mucosal) administration is desired, compositions may be ina form and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or adose having a particular particle size.

Compositions of the invention can contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing, especially ifthey are administered orally. The viscous compositions may be in theform of gels, lotions, ointments, creams and the like and will typicallycontain a sufficient amount of a thickening agent so that the viscosityis from about 2500 to 6500 cps, although more viscous compositions, evenup to 10,000 cps may be employed. Viscous compositions have a viscositypreferably of 2500 to 5000 cps, since above that range they become moredifficult to administer. However, above that range, the compositions canapproach solid or gelatin forms which are then easily administered as aswallowed pill for oral ingestion.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally, to animals, children, particularly small children,and others who may have difficulty swallowing a pill, tablet, capsule orthe like, or in multi-dose situations. Viscous compositions, on theother hand, can be formulated within the appropriate viscosity range toprovide longer contact periods with mucosa, such as the lining of thestomach or nasal mucosa.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form), or solid dosage form (e.g., whether thecomposition is to be formulated into a pill, tablet, capsule, caplet,time release form or liquid-filled form).

Solutions, suspensions and gels, normally contain a major amount ofwater (preferably purified water) in addition to the antigen, andoptional adjuvant. Minor amounts of other ingredients such as pHadjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents,buffering agents, preservatives, wetting agents, jelling agents, (e.g.,methylcellulose), colors and/or flavors may also be present. Thecompositions can be isotonic, i.e., it can have the same osmoticpressure as blood and lacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a harmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount whichwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions must be selected to be chemically inert with respect to thePspA antigen and optional adjuvant. This will present no problem tothose skilled in chemical and pharmaceutical principles, or problems canbe readily avoided by reference to standard texts or by simpleexperiments (not involving undue experimentation), from this disclosureand the documents cited herein.

The immunologically effective compositions of this invention areprepared by mixing the ingredients following generally acceptedprocedures. For example the selected components may be simply mixed in ablender, or other standard device to produce a concentrated mixturewhich may then be adjusted to the final concentration and viscosity bythe addition of water or thickening agent and possibly a buffer tocontrol pH or an additional solute to control tonicity. Generally the pHmay be from about 3 to 7.5.

Compositions can be administered in dosages and by techniques well knownto those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient or animal, and the composition form used foradministration (e.g., solid vs. liquid). Dosages for humans or otheranimals can be determined without undue experimentation by the skilledartisan from this disclosure, the documents cited herein and theExamples below (e.g., from the Examples involving mice).

Suitable regimes for initial administration and booster doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan from this disclosure, thedocuments cited herein and the Examples below.

The following Examples are provided for illustration and are not to beconsidered a limitation of the invention.

EXAMPLES Example 1 Identification of Sequence Homologies Between PspAs

Despite the described diversity of PspA strains, the nucleotide andamino acids sequences of the PspA molecule has been evaluated withrespect to whether any region(s) of conservation have been maintainedwhich could be of utility to vaccine development. The comparison of thenucleotide and amino acid sequences from multiple strains of PspArevealed that the primary sequence of the alpha helix has two regions ofrelative conservation and a region of extensive diversity betweenstrains. The two regions of diversity are comprised of the first,N-terminal, 60 amino acids of the alpha helix, and the last, C-terminal,100 amino acids of the alpha helix, as shown in FIGS. 1 and 2.

FIG. 1 shows the nucleotide sequences of the alpha helix and prolineregions of the pspA genes from Rx1 and EF 10197, both members of thesame family or clade, as compared to each other for regions of homology.This comparison was made used a Pustell DNA matrix analysis within theMacVector version 5.0.2 software, using a window of 30 nucleotides, aminimum percentage of homology of 70%, a hash value of 6, and a jumpvalue of 1. Points or lines in the graph indicate regions of homologybetween the two genes that meet the aforementioned criteria. The resultsdemonstrate homology in the portions of the genes encoding theN-terminal and C-terminal ends of the alpha helix region, as well as theproline region.

FIG. 2 shows the amino acid sequence comparison of the alpha helix andproline regions of the PspA proteins from Rx1 and EF10197, both membersof the same family or clade, as compared to each other for regions ofhomology. This comparison was made using a Pustell protein matrixanalysis within the MacVector version 5.0.2 software. The analysis wasdone using a window of 8 amino acids, a minimum percentage homology of70%, a hash value of 2, and the pam250 scoring matrix. Points or linesin the graph indicate regions of homology between the two proteins. Theresults demonstrate homology in the N-terminal and the C-terminal endsof the alpha helix region, as well as in the proline region.

The conserved region at the C-terminal end of the alpha helix regioncorrelated with a region demonstrated to contain protective epitopesthat were conserved between two strains.

Reasoning that the C-terminal region of the alpha helix region wascritical to vaccine development, the heterogeneity and family structureof amino acid sequences in this region was examined. The comparison ofthe amino acid sequences in this region between 26 strains of PspArevealed that the PspA strains could be grouped into 6 clades withgreater than 75% homology. These clades could be grouped into 4 familieswith greater than 500% homology. These data are shown in Tables 1 to 6,and FIGS. 3 to 8.

TABLE 1 Family/Clade List % AMINO ACID HO- MOLOGY HOMOLOGY TO CLADEWITHIN CON- FAMILY FAMILY CLADE STRAIN SENSUS Family 1 >50% Clade 1BG9739 96 (SEQ ID (SEQ ID NO:4) NO:1) DBL6A 98 (SEQ ID NO:5) L81905 94(SEQ ID NO:6) BG8743 87 (SEQ ID NO:7) AC94 88 (SEQ ID NO:9) BG6692 96(SEQ ID NO:2) BG8838 95 (SEQ ID NO:3) DBL1 88 (SEQ ID NO:8) Clade 2EF10197 89 (SEQ ID (SEQ ID NO:11) NO:10) RX1 92 (SEQ ID NO:12) WU2 87(SEQ ID NO:13) 0922134 99 (SEQ ID NO:16) DBL5 92 (SEQ ID NO:14) BG916379 (SEQ ID NO:17) EF6796 91 (SEQ ID NO:15) Family 2 >50% Clade 3 EF329697 (SEQ ID (SEQ ID NO:20) NO:18) AC122 96 (SEQ ID NO:19) BG8090 96 (SEQID NO:21) Family 3 >50% Clade 4 EF5668 92 (SEQ ID (SEQ ID NO:25) NO:22)BG7817 96 (SEQ ID NO:24) BG7561 89 (SEQ ID NO:26) BG11703 100 (SEQ IDNO:23) Clade 5 ATCC6303 100 (SEQ ID NO:27) Family 4 >50% Clade 6 BG6380100 (SEQ ID NO:28)

TABLE 2A Homology Between Clades-Matrix of Amino Acid SimilarityEstimates Between Clades Clade 1 Clade 2 Clade 3 Clade 4 Clade 5 Clade 6Clade 1 >75% Clade 2 >50% >75% Clade 3 <25% <20% >75% Clade 4<20% >30% >30% >75% Clade 5 <20% <20% >30% >50% >75% Clade 6 <10% <20%<10% <20% <20% >75%

TABLE 2B AA % sequence identities to PspA Clade Consensus % of AA thatdiffer Strain Name (Capsular from the Clade Con- % AA identity to CladeType) sensus Clade Consensus Clade 1 BG9739 (4) 4 96 DBL6A (6A) 2 98L81905 (4) 6 94 BG8743 (23) 13 87 AC94 (9) 12 88 BG6692 (33) 4 96 BG8838(6) 5 95 DBL1 (6B) 12 88 Clade 2 EF10197 (3) 10 89 RX1 (2) 8 92 WU2 (3)13 87 0922134 (23) 1 99 DBL5 (5) 8 92 BG9163 (6B) 21 79 EF6796 (6A) 9 91Clade 3 EF3296 (4) 1 97 AC122 (9) 2 96 BG8090 (19) 4 96 Clade 4 EF5668(4) 9 92 BG7817 (7) 4 96 BG7561 (15) 12 89 BG11703 (N.D) 0 100 Clade 5ATCC6303 (3) 0 100 Clade 6 BG6380 (37) 0 100 N.D = not determined

TABLE 3 Sequence identities to PspA Clade 1 Consensus # OF AA THATDIFFER FROM % AA IDENTITY TO THE CLADE CON- CLADE CLADE STRAIN NAMESENSUS CONSENSUS Clade 1 BG9739 4 96 DBL6A 2 98 L81905 6 94 BG8743 13 87AC94 12 88 BG6692 4 96 BG8838 5 95 DBL1 12 88

TABLE 4 Sequence identities to PspA Clade 2 consensus # OF AA THATDIFFER FROM % AA IDENTITY TO THE CLADE CON- CLADE CLADE STRAIN NAMESENSUS CONSENSUS Clade 2 EF10197 10 89 RX1 8 92 WU2 13 87 0922134 1 99DBL5 8 92 BG9163 21 79 EF6796 9 91 RCT123 3 97 RCT129 1 99 RCT135 0 100LXS200 0 100

TABLE 5 Sequence identities to PspA Clade 3 Consensus # OF AA THATDIFFER FROM % AA IDENTITY TO THE CLADE CON- CLADE CLADE STRAIN NAMESENSUS CONSENSUS Clade 3 EF3296 1 97 AC122 2 96 BG8090 4 96

TABLE 6 Sequence identities to PspA Clade 4 Consensus # OF AA THATDIFFER FROM % AA IDENTITY TO THE CLADE CON- CLADE CLADE STRAIN NAMESENSUS CONSENSUS Clade 4 EF5668 9 92 BG7817 4 96 BG7561 12 89 BG11703 0100

The immunological relevance of these families was demonstrated byserological analysis of S. pneumoniae strains with a large number ofmonoclonal antibodies made to several different PspAs. As shown in Table7, the pattern of reactions with strains in clades 3, 4, 5 and 6 ofmonoclonal antibodies generally correlated with the defined clade bysequence.

TABLE 7 Ab Reactions Clades 3-6 Anti-PspA Monoclonal Antibodies Made toEF5668(P56) Made to EF3296(P32) STRAIN CLADE* 263D12 263F6 264A4 264A11265E6 270B6 263B7 351G12 EF3296 3 X X X X X X X BG7140 X X X X XPMsv1281 X X X X X X VH1193 X X X X X X X EF5668 4 X BG7817 4 X BG7561 4X BG11703 4 X BG7736 X BG7813 X BG7915 X BG10717- X /30 ATCC6- 5 X 306BG7619 X BG7941 X BG13075- X /30 B06380 6 X indicates a positivereaction *clade was determined by amino acid sequences

Example 2 Competitive Inhibition of Anti-Rx1 Polyclonal Antibodies Withthe PspA Antigens of Different Strains

Competitive inhibition of anti-PARx1 binding to PARx1 antigen wasanalyzed using a BIAcore® sensory chip, coated with PARx1 antigen. Theresults are shown in FIG. 9. Rabbit polyclonal anti-PARx1 (1200 ng/ml)was allowed to react to the chip either alone, or in the presence ofincreasing concentration of PARx1 antigen (indicated by + in FIG. 9) orPA314 PspA antigen (indicated by squares in FIG. 9); the PA314 PspAantigen contains amino acids 96 to 314 of Rx1. The concentration ofuninhibited antibody able to bind to the PARx1 antigen on the sensorychip surface was measured using mass transport measurements on theBIAcore® instrument. The mouse monoclonal IgG anti-PspA antibody,P81-122F10.A11 was used as a standard for these measurements.

The results of these experiments indicated that the N-terminal conservedregion does not contain antigenic epitopes for the PspA response, andthat the conserved region at the C-terminal end of the alpha helixcontains cross-reactive and protective epitopes that are critical to thedevelopment of a broadly efficacious PspA vaccine. Further, FIG. 9demonstrates the lack of relevance of the first 60 amino acids of theN-terminal region of the alpha helix, as the PA314 PspA antigen used inthe competition assays above contains amino acids 96 to 314 of Rx1.

FIG. 10 shows the inhibition of PARx1 and PAEF5668 antigens. A BIA core®sensory chip was coated with PARx1 antigen and rabbit polyclonalanti-PARx1 (7 mM) was allowed to react to the chip either alone, or inthe presence of increasing concentration of PARx1 antigen (representedby squares in FIG. 10) or PAEF5668 antigen (represented by circles inFIG. 10). The concentration (mM) of these competitive antigens is shownon the X axis on a logarithmic scale, while the concentration (mM) ofuninhibited polyclonal antibody able to bind to the PARx1 antigen on thesensory chip was measured using mass transport measurements on theBIAcore® instrument, and is shown on the Y axis in FIG. 10.

As expected, the concentration of active, non-competitively inhibitedpolyclonal anti-PARx1 decreased as the concentrations of competitiveinhibitor increased. PARx1 antigen completely inhibited the polyclonalantibodies at sufficient concentrations of antigens in excess. ThePAEF5668 antigen has a maximal inhibition of 8.4%. The mouse monoclonalIgG anti-PspA antibody, P81-122F10. A11 was used as a standard forcalculating the concentrations of active polyclonal antibody in thisassay.

The results of the inhibition study by PARx1 and PABG6380 antigens isshown in FIG. 11. A BIAcore® sensory chip was coated with PARx1 antigenand rabbit polyclonal anti-PARx1(7 mM) was allowed to react to the chipeither alone, or in the presence of increasing concentration of PARx1antigen (represented by squares in FIG. 11), or PABG6380 antigens(represented by X's in FIG. 11). The concentration (mM) of thesecompetitive antigens is shown on the X axis on a logarithmic scale,while the concentration (mM) of uninhibited polyclonal antibody able tobind to the PARx1 antigen on the sensory chip was measured using masstransport measurements on the BIAcore® instrument, and is shown on the Yaxis in FIG. 11.

As expected, the concentration of active, non-competitively inhibitedpolyclonal anti-PARx1 decreased as the concentration of competitiveinhibitor increased. PARx1 antigen completely inhibited the polyclonalantibodies at sufficient concentrations of antigen in excess. ThePABG6380 antigen did not significantly inhibit the polyclonal antibodyreaction. The mouse monoclonal IgG anti-PspA antibody P81-122F10.A11 wasused as a standard for calculating the concentrations of activepolyclonal antibody in the assay.

Further, Table 8 shows the results of inhibition studies of polyclonalrabbit anti-Rx1 antibodies with representative strains of selectiveclades. As shown in the Table, anti-Rx1 antibodies inhibit clade 2effectively, but the inhibition of PspAs in clades which differ from thespecificity of the antibody itself is less effective.

TABLE 8 Inhibition of Polyclonal Rabbit anti-Rx1 Antibodies (Inhibitionof Anti-Clade 2 Antibody Reactivity Clade 1 2 2 2 4 6 — Strain BG9739RX1 R36A WU2 EF5668 BG6380 JY1119* Antigen BG9739/n PARx1 R36A/n WU2/nPAEF5668 PABG6380 JY1119/n Name Antigen native recom.** native nativerecom.** recom.** native Type % 35.4 100.0 100.0 91.4 8.4 0.0 0.0Inhibition *JY1119 is an engineered PspA loss mutant and is used in thisassay as a negative control **recom. = recombinant

Example 3 Competitive Inhibition of Clade Specific Anti-PspA PolyclonalAntibodies With PspA Antigens from Different Clades

Rabbit polyclonal antiserum was raised against recombinantly expressedPspA antigens from representatives of the six clades. BG9739 was chosenas the representative of clade 1, Rx1 was the representative of clade 2,EF3296 was the representative of clade 3, EF5668 was the representativeof clade 4, ATCC 6303 was the representative of clade 5 and BG6380 wasthe representative of clade 6.

Recombinant PspA antigens from each of the clades were each covalentlylinked to individual BIAcore sensory chips. Next, clade-specificpolyclonal anti-PspA antibodies were allowed to react with each chip,either alone or in the presence of increasing concentration of eachrecombinant PspA antigen. The active concentration of anti-PspAantibodies that could bind to the PspA on the chip were determined usingmass transport measurement. By comparing concentrations of the activeantibody in uninhibit reactions and in reactions inhibited by theaddition of soluble PspA strains was determined. These results areillustrated as estimated cross-reactivities in Table 9.

Cross-reactivity analyses were also performed using recombinant PspAantigens to coat BIAcore chips and the clade-specific polyclonalantisera described above, inhibited with native PspA antigens fromrepresentative clades. The results of the estimated cross-reactivitiesare shown in Table 10, and compare favorably to the results obtainedwith the results shown in Table 9. In each case, representatives of thesame clade demonstrate the greatest cross-reactivity. Representatives ofdifferent clades within the same family demonstrate moderatecross-reactivity, and representatives of different families show theleast cross-reactivity.

TABLE 9 Cross-Reacting PspA Antigens Family 1 2 3 4 Clade 1 2 3 4 5 6Rabbit Anti-PspA Strain Strain Family Clade Strain BG9739 Rx1 EF3296EF5668 ATCC 6303 BG6380 1 1 BG9739 100 23 0 0 0 1 2 Rx1 34 100 0 5 0 3 23 EF3296 0 0 100 0 0 2 3 4 EF5668 4 5 10 100 67 1 5 ATCC 6303 0 0 7 86100 0 4 6 BG6380 0 0 4 0 0 100

Example 4 Serotype Analysis of S. pneumoniae strains by ELISA

Rabbit polyclonal antiserum was raised against recombinantly expressedPspA antigens from representatives of the six clades. BG739 was chosenas the representative of clade 1, Rx1 was the representative of clade 2,EF3296 was the representative of clade 3, EF5668 was the representativeof clade 4, ATCC 6303 was the representative of clade 5 and BG6380 wasthe representative of clade 6.

Each of the antisera were first normalized to contain roughly equivalentspecific activities. Then, sequential 1.5 fold dilutions o the antiserawere reacted with ELISA plates coated with cetavalon lysates ofrepresentatives of each clade. As a negative control, the pspA gene of aS. pneumoniae strain was “knocked out”, i.e., rendered inactive by arecombination event, and cetavalon lysates of this knock out strain wereused to coat ELISA plate wells. Next, each well was incubated with goatanti-rabbit IgG-alkaline phosphatase conjugates (Kierkegaard and Perry,Gaithersburg, Md.) and washed, then p-Nitrophenyl Phosphate (SigmaDiagnostics, St. Louis) was added. Results of the colorimetric reactionswere read at 405 nm.

Mixtures of antisera were prepared, combining equal specific activitiesof anti-Rx1, EF3296 and EF5668 in one cocktail, and anti-Rx1, EF3296,EF5668 and BG6380 in a second cocktail. These cocktails containedantibodies which reacted with clades 2, 3 and 4 or 2, 3, 4 and 6 andwere used to approximate the ability of various vaccine combinations toconfer broad inmunity. Mixtures of these antibodies reacted well witheach of the S. pneumoniae strains tested, demonstrating thatcombinations of vaccines based on the clade definitions of the presentinvention should confer immunity against a broad range of S. pneumoniaeisolates.

Polyclonal antibodies to PspAs from individual clades demonstratedlittle cross-reactivity with representatives of other fanilies.Significant cross-reactivity was observed between strains of clades 1and 2 and between strains of clades 4 and 5. This observation isconsistent with the grouping of these clades into families 1 and 3,respectively. Each strain could be serotyped and placed within a definedfamily or clade of PspA based on reactivity with polyclonal anti-PspA.

A total of 437 S. pneumoniae strains from the United States and Europewere evaluated by the polyclonal anti-PSA ELISA method. The results ofthis analysis are shown in Table 11. Approximately 36% of all strainsexamined were serotyped as clade 2, 22% as clade 3 and 23% as clade 4. Avaccine comprised of PspAs from these three clades alone would covergreater than 80% of the S. pneumoniae% of these strains could beserotyped into one of the six clades, again demonstrating the potentialfor a finite number of vaccine components based on clade-specific PspAsto confer broad immunity against infection caused by S. pneumoniae. Infact, based on the high degree of cross-reactivity within families, avaccine composition comprised of a single representative member of eachfamily should confer such immunity.

Having thus described in detail certain preferred embodiments of thepresent invention, it is to be understood that the invention defined bythe appended claims is not to be limited by particular details set forthin the above description, as many apparent variations thereof arepossible without departing from the spirit or scope thereof.

1. Center of Disease Control. 19484. Pneumococcal polysaccharide vaccineusage, United States, MMWR 33: 273-276, 281.

2. Mufson, M. A., G. Oley and D. Hughey, 1982. Pneumococcal disease in amedium-sized community in the United States. JAMA 248: 1486-1489.

3. Hook, E. W., C. A. Horton and D. R. Schaberg. 1983. Failure ofintensive care unit support to influence mortality from pneumococcalbacteremia. JAMA 249: 1055-1057.

4. Breiman, R. F., J. S. Spika, V. J. Navarro, P. M. Darden and C. P.Darby. 1990. Pneumococcal bacteremia in Charleston County, SouthCarolina. Arch. Intern. Med. 150: 1401-1405.

5. Afessa, B., W. L. Greaves and W. R. Frederick. 1995. Pneumococcalbacterimia in adults: a 14-year experience in an inner-city universityhospital. Clin. Infec. Diseases 21: 345-351.

6. Fang, G. D., M. Fine, J. Orloff, D. Arisumi, V. L. Yu, W. Kapoor, J.T., Grayston, S. P. Wang, R. Kohler, R. R. Muder and et al. 1990. Newand emerging etiologies for community-acquired pneumonia withimplications for therapy. A prospective multicenter study of 359 cases.Medicine (Baltimore) 69: 307-316.

7. Marrie, T. J., H. Durant and L. Yates. 1989. Community-acquiredpneumonia requiring hospitalization: 5-year prospective study. Rev.Infect. Dis. 11: 586-599.

8. Torres, A., J. Serra-Batlles, A. Ferrer, P. Jimeniz, R. Celis, E.Cobo and R. Rodriquez-Roisin. 1991. Severe community-acquired pneumonia.Epidemiology and prognostic factors. Am Rev Respir Dis. 144: 312-318.

9. Bluestone, C. D., J. S. Stephenson and L. M. Martin. 1992. Ten-yearreview of otitis media pathogens. Pediatr. Infect. Dis. J. 11: S7-11.

10. Teele, D. W., J. O. Klein, B. Rosner and G. B. O. M. S. Group. 1989.Epidemiology of otitis media during the first seven years of life ofchildren in greater Boston: a prospective cohort study. J. Infect. Dis.160: 83-94.

11. Schutze, G. E., S. L. Kaplan and R. F. Jacobs. 1994. Resistantpneumococcus: A worldwide problem. Infection 22:233-237.

12. Privitera, G. 1994. Penicillin resistance among Streptococcuspneumoniae in Europe. Diagnostic Microbiology and Infectious Disease 19:157-161.

13. Bizzozero, O. G. Jr. and V. T. Andriole. 1969.Tetracycline-resistant pneumococcal infection. Incidence, clinicalpresentation and laboratory evaluation. Arch Intern Med. 123: 388-393.

14. Workman, M. R., M. Layton, M. Hussein, J. Philpott-Howard and R. C.George. 1993. Nasal carriage of penicillin-resistant pneumococcus insickle cell patients (letter). Lancet 342: 746-747.

15. Koornhof, H. J., A. Wasas and K. Klugman. 1992. Antimicrobialresistance in Streptococcus pneumoniae: a South African perspective.Clin. Infect. Dis. 15: 84-94.

16. Dagan, R., P. Yagupsky, A. Goldbart, A. Wasas and K. Klugman. 1994.Increasing prevalence of penicillin-resistant pneumococcal infections inchildren in southern Israel: implications for future immunizationpolicies. Pediatr. Infect. Dis. J. 13: 782-786.

17. Reichler, M. R., J. Rakovsky, A. Sobotova, M. Slacikova, B.Hlavacova, B. Hill, L. Krajcikova, P. Tarina, R. R. Facklam and R. F.Breiman. 1995. Multiple antimicrobial resistance of pneumococci inchildren with otitis media, bacteremia, and meningitis in Slovakia. J.Infect. Dis. 171: 1491-1496.

18. Freidland, I. R., S. Shelton, M. Paris, S. Rinderknecht, S. Ehrett,K. Krisher, and G. H. McCracken, Jr., 1993. Dilemmas in diagnosis andmanagement of cephalosporin-resistant Streptococcus pneumoniaemeningitis. Pediatr. Infect. Dis. J. 12: 196-200.

19. Fedson, D. S., and D. M. Musher. 1994. Pneumococcal Vaccine. InVaccines. S. A. Plotkin and J. E. A. Montimer, Eds. W. B. Saunders Co.,Philadelphia, Pa., p. 517-564.

20. Takala, A. K., J. Eskola, M. Leinonen, H. Kayhty, A. Nissinen, E.Pekkanen and P. H. Makela. 1991. Reduction of oropharyngeal carriage ofHaemophilus influenzae type b (Hib) in children immunized with an Hibconjugate vaccine. J. Infect. Dis. 164: 982-986.

21. Takala, A. K., M. Santosham, J. Almeido-Hill, M. Wolff, W. Newcomer,R. Reid, H. Kayhty, E. Esko and P. H. Makela 1993. Vaccination withHaemophilus influenzae type b meningococcal protein conjugate vaccinereduces oropharyngeal carriage of Haemophilus influenzae type b amongAmerican Indian children. Pediatr. Infect. Dis. J. 12: 593-599.

22. Ward, J., J. M. Lieberman and S. L. Cochi. 1994. Haemophilusinfluenzae vaccines. In Vaccines. S. A. Plotkin and J. E. A. Montimer,Eds. W. B. Saunders Co., Philadelphia, Pa., p. 337-386.

23. Murphy, T. V., P. Pastor, F. Medley, M. T. Osterholm, and D. M.Cranoff. 1993. Decreased Haemophilus colonization in children vaccinatedwith Haemophilus influenzae type b conjugate vaccine. J. Pediatr. 122:517-523.

24. Mohle-Boetani, J. C., G. Ajello, E. Breneman, K. A., Deaver, C.Harvey, B. D. Plikaytis, M. M. Farley, D. S. Stephens and J. D. Wenger.1993. Carriage of Haemophilus influenzae type b in children afterwidespread vaccination with conjugate Haemophilus influenzae type bvaccines. Pediatr. Infect. Dis. J. 12: 589-593.

25. Watson, D. A. and D. M. Musher. 1990. Interruption of capsuleproduction in Streptococcus pneumonia serotype 3 by insertion oftransposon Tn916. Infect. Immun. 58: 135-138.

26. Avery, O. T. and R. Dubos. 1931. The protective action of specificenzyme against type III pneumococcus infection in mice. J. Exp. Med. 54:73-89.

27. Alonso DeVelasco, E., A. F. M. Verheul, J. Verhoef and H. Snippe.1995. Streptococcus pneumoniae: virulence factors, pathogenesis andvaccines. Microbiological Reviews 59: 591-603.

28. Butler, J. C., R. F. Breiman, J. F. Campbell, H. B. Lipman, C. V.Broome and R. R. Facklam. 1993. Pneumococcal polysaccharide vaccineefficacy. An evaluation of current recommendations. JAMA 270: 1826-1831.

29. Hirschmann, J. V., and B. A. Lipsky. 1994. The pneumococcal vaccineafter 15 years of use. Arch Intern Med. 154: 373-377.

30. Briles, D. E., J. Yother and L. S. McDaniel. 1988. Role ofpneumococcal surface protein A in the virulence of Streptococcuspneumoniae. Rev. Infect. Dis. 10: S372-4.

31. Talkington, D. F., D. C. Voellinger, L. S. McDaniel and D. E.Briles. 1992. Analysis of pneumococcal PspA microheterogeneity inSDS-polyacrylaminde gels and the association of PspA with the cellmembrane. Microb. Pathogen. 13: 343-355.

32. Yother, J. and D. E. Briles. 1992. Structural properties andevolutionary relationships of PspA, a surface protein of Streptococcuspneumoniae, as revealed by sequence analysis. J. Bacteriol. 174:601-609.

33. Yother, J. and J. M. White. 1994. Novel surface attachment mechanismof the Streptococcus pneumoniae protein PspA. J. Bacteriol. 176:2976-85.

34. McDaniel, L. S., B. A. Ralph, D. O. McDaniel and D. E. Briles. 1994.Localization of protection-eliciting epitopes of PspA of Streptococcuspneumoniae between amino acids residues 192 and 260. Microb. Pathog. 17:323-337.

35. Ralph, B. A., D. E. Briles and L. S. McDaniel. 1994. Cross-reactiveprotection eliciting epitopes of pneumococcal surface protein A. Ann N YAcad. Sci. 730: 361-3.

36. Waltman, W. D., L. S. McDaniel, B. Andersson, L. Bland, B. M. Gray,C. S. Eden and D. E. Briles. 1988. Protein serotyping of Streptococcuspneumoniae based on reactivity to six monoclonal antibodies. Microb.Pathog. 5: 159-67.

28 1 98 PRT Streptococcus pneumoniae 1 Leu Lys Glu Ile Asp Glu Ser AspSer Glu Asp Tyr Val Lys Glu Gly 1 5 10 15 Leu Arg Ala Pro Leu Gln SerGlu Leu Asp Ala Lys Gln Ala Lys Leu 20 25 30 Ser Lys Leu Glu Glu Leu SerAsp Lys Ile Asp Glu Leu Asp Ala Glu 35 40 45 Ile Ala Lys Leu Glu Lys AspVal Glu Asp Phe Lys Asn Ser Asp Gly 50 55 60 Glu Gln Ala Gln Tyr Leu AlaAla Ala Glu Glu Asp Leu Ala Lys Lys 65 70 75 80 Ala Glu Leu Glu Lys ThrGlu Ala Asp Leu Lys Lys Ala Val His Glu 85 90 95 Pro Glu 2 100 PRTStreptococcus pneumoniae 2 Leu Lys Glu Ile Asp Glu Ser Asp Ser Glu AspTyr Val Lys Glu Gly 1 5 10 15 Leu Arg Ala Pro Leu Gln Ser Glu Leu AspAla Lys Gln Ala Lys Leu 20 25 30 Ser Lys Leu Glu Glu Leu Ser Asp Lys IleAsp Glu Leu Asp Ala Glu 35 40 45 Ile Ala Lys Leu Glu Lys Asn Val Glu AspPhe Lys Asn Ser Asn Gly 50 55 60 Glu Gln Ala Glu Gln Tyr Arg Ala Ala AlaGlu Glu Asp Leu Ala Ala 65 70 75 80 Lys Gln Ala Glu Leu Glu Lys Thr GluAla Asp Leu Lys Lys Ala Val 85 90 95 His Glu Pro Glu 100 3 100 PRTStreptococcus pneumoniae 3 Leu Lys Glu Ile Asp Glu Ser Asp Ser Glu AspTyr Val Lys Glu Gly 1 5 10 15 Leu Arg Ala Pro Leu Gln Ser Glu Leu AspAla Lys Gln Ala Lys Leu 20 25 30 Ser Lys Leu Glu Glu Leu Ser Asp Lys IleAsp Glu Leu Asp Ala Glu 35 40 45 Ile Ala Lys Leu Glu Lys Asn Val Glu AspPhe Lys Asn Ser Asn Gly 50 55 60 Glu Glu Ala Glu Gln Tyr Arg Ala Ala AlaGly Glu Asp Leu Ala Ala 65 70 75 80 Lys Gln Ala Glu Leu Glu Lys Thr GluAla Asp Leu Lys Lys Ala Val 85 90 95 His Glu Pro Glu 100 4 100 PRTStreptococcus pneumoniae 4 Leu Lys Glu Ile Asp Glu Ser Asp Ser Glu AspTyr Val Lys Glu Gly 1 5 10 15 Glu Arg Ala Pro Leu Gln Ser Glu Leu AspAla Lys Gln Ala Lys Leu 20 25 30 Ser Lys Leu Glu Glu Leu Ser Asp Lys IleAsp Glu Leu Asp Ala Glu 35 40 45 Ile Ala Lys Leu Glu Lys Asp Val Glu AspPhe Lys Asn Ser Asp Gly 50 55 60 Glu Gln Ala Gly Gln Tyr Leu Ala Ala AlaGly Glu Asp Leu Ile Ala 65 70 75 80 Lys Lys Ala Glu Leu Glu Lys Ala GluAla Asp Leu Lys Lys Ala Val 85 90 95 Asp Glu Pro Glu 100 5 100 PRTStreptococcus pneumoniae 5 Leu Lys Glu Ile Asp Glu Ser Asp Ser Glu AspTyr Val Lys Glu Gly 1 5 10 15 Glu Arg Ala Pro Leu Gln Ser Glu Leu AspAla Lys Gln Ala Lys Leu 20 25 30 Ser Lys Leu Glu Glu Leu Ser Asp Lys IleAsp Glu Leu Asp Ala Glu 35 40 45 Ile Ala Lys Leu Glu Lys Asp Val Glu AspPhe Lys Asn Ser Asp Gly 50 55 60 Glu Gln Ala Gly Gln Tyr Leu Ala Ala AlaGlu Glu Asp Leu Ile Ala 65 70 75 80 Lys Lys Ala Glu Leu Glu Gln Thr GluAla Asp Leu Lys Lys Ala Val 85 90 95 His Glu Pro Glu 100 6 100 PRTStreptococcus pneumoniae UNSURE (1)..(100) amino acid ′Xaa′ can be anyamino acid 6 Leu Lys Glu Ile Asp Glu Ser Asp Ser Glu Asp Tyr Val Lys GluGly 1 5 10 15 Glu Arg Ala Pro Leu Gln Ser Glu Leu Asp Ala Lys Gln AlaLys Leu 20 25 30 Ser Lys Leu Glu Glu Xaa Ser Asp Lys Xaa Asp Glu Leu AspAla Glu 35 40 45 Ile Ala Lys Leu Glu Lys Asp Val Glu Asp Phe Lys Asn SerAsp Gly 50 55 60 Glu Gln Ala Gly Gln Tyr Leu Ala Ala Ala Glu Glu Asp LeuIle Ala 65 70 75 80 Lys Lys Ala Xaa Leu Glu Lys Ala Glu Ala Asp Leu LysLys Ala Val 85 90 95 Asp Glu Pro Glu 100 7 100 PRT Streptococcuspneumoniae UNSURE (1)..(100) amino acid ′Xaa′ can be any amino acid 7Leu Lys Glu Ile Asp Glu Ser Asp Ser Glu Asp Tyr Glu Lys Glu Gly 1 5 1015 Leu Arg Ala Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu 20 2530 Ser Lys Leu Asp Glu Xaa Ser Asp Lys Xaa Asp Glu Leu Asp Ala Glu 35 4045 Ile Ala Lys Leu Glu Lys Asp Val Gly Asp Phe Pro Asn Ser Asp Gly 50 5560 Glu Gln Ala Gly Gln Tyr Leu Val Ala Ala Glu Lys Asp Leu Asp Ala 65 7075 80 Lys Glu Ala Glu Leu Gly Asn Thr Gly Ala Asp Leu Lys Lys Ala Val 8590 95 Asp Glu Pro Glu 100 8 100 PRT Streptococcus pneumoniae 8 Leu LysGly Ile Asp Glu Ser Asp Ser Glu Asp Tyr Val Lys Glu Gly 1 5 10 15 LeuArg Ala Pro Leu Gln Ser Glu Leu Asp Ala Lys Arg Thr Lys Leu 20 25 30 SerThr Leu Glu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu 35 40 45 IlePro Lys Leu Glu Lys Asn Val Glu Tyr Phe Lys Leu Thr Asp Ala 50 55 60 GluGln Thr Glu Gln Tyr Leu Ala Ala Ala Glu Lys Asp Leu Ala Asp 65 70 75 80Lys Lys Ala Glu Leu Glu Lys Thr Glu Ala Asp Leu Lys Lys Ala Val 85 90 95His Glu Pro Glu 100 9 101 PRT Streptococcus pneumoniae 9 Leu Lys Glu IleAsp Glu Ser Asp Ser Glu Asp Tyr Val Lys Glu Gly 1 5 10 15 Leu Arg ValPro Leu Gln Ser Glu Leu Asp Val Lys Gln Ala Lys Leu 20 25 30 Leu Lys LeuGlu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu 35 40 45 Ile Ala LysAsn Leu Lys Lys Asp Val Glu Asp Phe Gln Asn Ser Gly 50 55 60 Gly Gly TyrSer Ala Leu Tyr Leu Glu Ala Ala Glu Lys Asp Leu Val 65 70 75 80 Ala LysLys Ala Glu Leu Glu Lys Thr Glu Ala Asp Leu Lys Lys Ala 85 90 95 Val HisGlu Pro Glu 100 10 100 PRT Streptococcus pneumoniae 10 Leu Lys Glu IleAsp Glu Ser Asp Ser Glu Asp Tyr Ala Lys Glu Gly 1 5 10 15 Phe Arg AlaPro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu 20 25 30 Ser Lys LeuGlu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu 35 40 45 Ile Ala LysLeu Glu Cys Val Gln Leu Lys Asp Ala Glu Gly Asn Asn 50 55 60 Asn Val GluAla Tyr Phe Lys Glu Gly Leu Glu Lys Thr Thr Ala Glu 65 70 75 80 Lys LysAla Glu Leu Glu Lys Ala Glu Ala Asp Leu Lys Lys Ala Val 85 90 95 Asp GluPro Glu 100 11 99 PRT Streptococcus pneumoniae 11 Leu Lys Glu Ile AspGlu Ser Asp Ser Glu Asp Tyr Val Lys Glu Gly 1 5 10 15 Phe Arg Ala ProLeu Gln Ser Glu Leu Asp Ala Lys Gln Ala Lys Leu 20 25 30 Ser Lys Leu GluGlu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu 35 40 45 Ile Ala Lys LeuGlu Asp Gln Leu Lys Ala Ala Glu Glu Asn Asn Asn 50 55 60 Val Glu Asp TyrPhe Lys Glu Gly Leu Glu Lys Thr Ile Ala Ala Lys 65 70 75 80 Lys Ala GluLeu Glu Lys Thr Glu Ala Asp Leu Lys Lys Ala Val Asn 85 90 95 Glu Pro Glu12 100 PRT Streptococcus pneumoniae 12 Leu Lys Glu Ile Asp Glu Ser GluSer Glu Asp Tyr Ala Lys Glu Gly 1 5 10 15 Phe Arg Ala Pro Leu Gln SerLys Leu Asp Ala Lys Gln Ala Lys Leu 20 25 30 Ser Lys Leu Glu Glu Leu SerAsp Lys Ile Asp Glu Leu Asp Ala Glu 35 40 45 Ile Ala Lys Leu Glu Asp GlnLeu Lys Lys Ala Ala Glu Glu Asn Asn 50 55 60 Asn Val Glu Asp Tyr Phe LysGlu Gly Leu Glu Lys Thr Ile Ala Ala 65 70 75 80 Lys Lys Ala Glu Leu GluLys Thr Glu Ala Asp Leu Lys Lys Ala Val 85 90 95 Asn Glu Pro Glu 100 1399 PRT Streptococcus pneumoniae 13 Leu Lys Glu Ile Asp Glu Ser Glu SerGlu Asp Tyr Ala Lys Glu Gly 1 5 10 15 Phe Arg Ala Pro Leu His Ser LysLeu Asp Ala Lys Gln Ala Lys Leu 20 25 30 Ser Lys Leu Glu Glu Leu Ser AspLys Ile Asp Glu Leu Asp Ala Glu 35 40 45 Ile Ala Lys Leu Glu Asp Gln LeuLys Ala Val Glu Glu Asn Asn Asn 50 55 60 Val Glu Asp Tyr Ser Thr Glu GlyLeu Glu Lys Thr Ile Ala Ala Lys 65 70 75 80 Lys Thr Glu Leu Glu Lys ThrGlu Ala Asp Leu Lys Lys Ala Val Asn 85 90 95 Glu Pro Glu 14 99 PRTStreptococcus pneumoniae UNSURE (1)..(99) amino acid ′Xaa′ can be anyamino acid 14 Leu Lys Asp Ile Asp Glu Ser Asp Ser Glu Asp Tyr Ala LysGlu Gly 1 5 10 15 Glu Arg Ala Pro Leu Gln Ser Glu Leu Asp Thr Lys LysAla Lys Leu 20 25 30 Leu Lys Leu Glu Glu Leu Ser Gly Lys Ile Glu Glu LeuAsp Ala Glu 35 40 45 Ile Xaa Glu Leu Glu Val Gln Leu Lys Asp Ala Glu GlyAsn Asn Asn 50 55 60 Val Glu Ala Tyr Phe Lys Glu Gly Leu Glu Lys Thr ThrAla Glu Lys 65 70 75 80 Lys Ala Glu Leu Glu Lys Ala Glu Ala Asp Leu LysLys Ala Val Asp 85 90 95 Glu Pro Glu 15 99 PRT Streptococcus pneumoniae15 Leu Glu Glu Ile Asn Glu Ser Asp Ser Glu Asp Tyr Ala Lys Glu Gly 1 510 15 Phe Arg Ala Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu 2025 30 Leu Lys Leu Glu Glu Leu Ser Gly Lys Ile Glu Glu Leu Asp Ala Glu 3540 45 Ile Ala Glu Leu Glu Val Gln Leu Lys Asp Ala Glu Gly Asn Asn Asn 5055 60 Val Glu Ala Tyr Phe Lys Glu Gly Leu Glu Lys Thr Thr Ala Glu Lys 6570 75 80 Lys Ala Glu Leu Glu Lys Ala Glu Ala Asp Leu Lys Lys Ala Val Asp85 90 95 Glu Pro Glu 16 99 PRT Streptococcus pneumoniae 16 Leu Lys GluIle Asp Glu Ser Asp Ser Glu Asp Tyr Leu Lys Glu Gly 1 5 10 15 Glu ArgAla Pro Leu Gln Ser Lys Leu Asp Thr Lys Lys Ala Lys Leu 20 25 30 Ser LysLeu Glu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu 35 40 45 Ile AlaLys Leu Glu Val Gln Leu Lys Asp Ala Glu Gly Asn Asn Asn 50 55 60 Val GluAla Tyr Phe Lys Glu Gly Leu Glu Lys Thr Thr Ala Glu Lys 65 70 75 80 LysAla Glu Leu Glu Lys Ala Glu Ala Asp Leu Lys Lys Ala Val Asp 85 90 95 GluPro Glu 17 99 PRT Streptococcus pneumoniae UNSURE (1)..(99) amino acid′Xaa′ can be any amino acid 17 Pro Lys Arg Ile Met Ser Leu Ser Gln LysVal Xaa Leu Lys Xaa Val 1 5 10 15 Cys Arg Ala Pro Leu Gln Ser Lys LeuAsp Ala Gln Lys Ala Glu Leu 20 25 30 Leu Lys Leu Glu Glu Leu Ser Gly LysIle Lys Glu Leu Asp Ala Glu 35 40 45 Ile Ala Glu Leu Glu Val Gln Leu LysAsp Ala Glu Gly Asn Asn Asn 50 55 60 Val Glu Ala Tyr Phe Lys Glu Gly LeuGlu Lys Thr Thr Ala Glu Lys 65 70 75 80 Lys Ala Glu Leu Glu Xaa Ala XaaAla Asp Leu Lys Lys Ala Val Asp 85 90 95 Glu Pro Glu 18 102 PRTStreptococcus pneumoniae 18 Leu Ala Lys Lys Gln Thr Glu Leu Glu Lys LeuLeu Asp Leu Asp Pro 1 5 10 15 Glu Gly Lys Thr Gln Asp Glu Leu Asp LysGlu Ala Glu Ala Glu Leu 20 25 30 Asp Lys Lys Ala Asp Glu Leu Pro Asn LysVal Ala Asp Leu Glu Lys 35 40 45 Glu Ile Ser Asn Leu Glu Ile Leu Leu GlyGly Ala Asp Ser Glu Asp 50 55 60 Asp Thr Ala Ala Leu Pro Asn Lys Leu AlaThr Lys Lys Ala Glu Leu 65 70 75 80 Glu Lys Thr Gln Lys Glu Leu Asp AlaAla Leu Asn Glu Leu Gly Pro 85 90 95 Asp Gly Asp Glu Glu Glu 100 19 80PRT Streptococcus pneumoniae 19 Leu Asp Lys Glu Ala Gly Glu Ala Glu LeuAsp Lys Lys Ala Asp Gly 1 5 10 15 Leu Pro Asn Lys Val Ser Asp Leu GluLys Glu Ile Ser Asn Leu Glu 20 25 30 Ile Leu Leu Gly Gly Ala Asp Ser GluAsp Asp Thr Ala Ala Leu Pro 35 40 45 Asn Lys Leu Ala Thr Lys Lys Ala GluLeu Glu Lys Thr Gln Lys Glu 50 55 60 Leu Asp Ala Ala Leu Asn Glu Leu GlyPro Asp Gly Asp Glu Glu Glu 65 70 75 80 20 104 PRT Streptococcuspneumoniae 20 Leu Ala Lys Lys Gln Thr Glu Leu Glu Lys Leu Leu Asp SerLeu Asp 1 5 10 15 Pro Glu Gly Lys Thr Gln Asp Glu Leu Asp Lys Glu AlaGlu Glu Ala 20 25 30 Glu Leu Asp Lys Lys Ala Asp Glu Leu Pro Asn Lys ValAla Asp Leu 35 40 45 Glu Lys Glu Ile Ser Asn Leu Glu Ile Leu Leu Gly GlyAla Asp Ser 50 55 60 Glu Asp Asp Thr Ala Ala Leu Pro Asn Lys Leu Ala ThrLys Lys Ala 65 70 75 80 Glu Leu Glu Lys Thr Gln Lys Glu Leu Asp Ala AlaLeu Asn Glu Leu 85 90 95 Gly Pro Asp Gly Asp Glu Glu Glu 100 21 104 PRTStreptococcus pneumoniae 21 Leu Ala Lys Lys Gln Thr Glu Leu Glu Lys LeuLeu Asp Asn Leu Asp 1 5 10 15 Pro Glu Gly Lys Thr Gln Asp Glu Leu AspLys Glu Ala Ala Glu Ala 20 25 30 Glu Leu Asp Lys Lys Ala Asp Glu Leu ProAsn Lys Val Ala Asp Leu 35 40 45 Glu Lys Glu Ile Ser Asn Leu Glu Ile LeuLeu Gly Gly Ala Asp Pro 50 55 60 Glu Asp Asp Thr Ala Ala Leu Pro Asn LysLeu Ala Thr Lys Lys Ala 65 70 75 80 Glu Leu Glu Lys Thr Gln Lys Glu LeuAsp Ala Ala Leu Asn Glu Leu 85 90 95 Gly Pro Asp Gly Asp Glu Glu Glu 10022 106 PRT Streptococcus pneumoniae 22 Leu Glu Lys Ala Glu Ala Glu LeuGlu Asn Leu Leu Ser Thr Leu Asp 1 5 10 15 Pro Glu Gly Lys Thr Gln AspGlu Leu Asp Lys Glu Ala Ala Glu Ala 20 25 30 Glu Leu Asn Lys Lys Val GluAla Leu Pro Asn Gln Val Glu Leu Glu 35 40 45 Glu Glu Leu Ser Lys Leu GluAsp Asn Leu Lys Asp Ala Glu Thr Asn 50 55 60 Val Glu Asp Tyr Ile Lys GluGly Leu Glu Glu Ala Ile Ala Thr Lys 65 70 75 80 Gln Ala Glu Leu Glu LysThr Pro Lys Glu Leu Asp Ala Ala Leu Asn 85 90 95 Glu Leu Gly Pro Asp GlyAsp Glu Glu Glu 100 105 23 108 PRT Streptococcus pneumoniae 23 Leu GluLys Ala Glu Ala Glu Leu Glu Asn Leu Leu Ser Thr Leu Asp 1 5 10 15 ProGlu Gly Lys Thr Gln Asp Glu Leu Asp Lys Glu Ala Ala Glu Ala 20 25 30 GluLeu Asn Lys Lys Val Glu Ala Leu Pro Asn Gln Val Ser Glu Leu 35 40 45 GluGlu Glu Leu Ser Lys Leu Glu Asp Asn Leu Lys Asp Ala Glu Thr 50 55 60 AsnAsn Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu Glu Ala Ile Ala 65 70 75 80Thr Lys Gln Ala Glu Leu Glu Lys Thr Pro Lys Glu Leu Asp Ala Ala 85 90 95Leu Asn Glu Leu Gly Pro Asp Gly Asp Glu Glu Glu 100 105 24 108 PRTStreptococcus pneumoniae 24 Leu Glu Lys Ala Gly Ala Gly Leu Glu Asn LeuLeu Ser Thr Leu Asp 1 5 10 15 Pro Glu Gly Lys Thr Gln Asp Glu Leu AspLys Glu Ala Ala Glu Ala 20 25 30 Glu Leu Asn Lys Lys Val Glu Ala Leu ProAsn Gln Val Ala Glu Leu 35 40 45 Glu Glu Glu Leu Ser Lys Leu Glu Asp AsnLeu Lys Asp Ala Glu Thr 50 55 60 Asn His Val Glu Asp Tyr Ile Lys Glu GlyLeu Glu Glu Ala Ile Ala 65 70 75 80 Thr Lys Gln Ala Glu Leu Glu Lys ThrPro Lys Glu Leu Asp Ala Ala 85 90 95 Leu Asn Glu Leu Gly Pro Asp Gly AspGlu Glu Glu 100 105 25 108 PRT Streptococcus pneumoniae 25 Leu Glu AspAla Glu Leu Glu Leu Glu Lys Val Leu Ala Thr Leu Asp 1 5 10 15 Pro GluGly Lys Thr Gln Asp Glu Leu Asp Lys Glu Ala Ala Glu Ala 20 25 30 Glu LeuAsn Glu Lys Val Glu Ala Leu Gln Asn Gln Val Ala Glu Leu 35 40 45 Glu GluGlu Leu Ser Lys Leu Glu Asp Asn Leu Lys Asp Ala Glu Thr 50 55 60 Asn AsnVal Glu Asp Tyr Ile Lys Glu Gly Leu Glu Glu Ala Ile Ala 65 70 75 80 ThrLys Lys Ala Glu Leu Glu Lys Thr Gln Lys Glu Leu Asp Ala Ala 85 90 95 LeuAsn Glu Leu Gly Pro Asp Gly Asp Glu Glu Glu 100 105 26 108 PRTStreptococcus pneumoniae UNSURE (1)..(108) amino acid ′Xaa′ can be anyamino acid 26 Leu Glu Lys Ala Glu Ala Glu Leu Glu Asn Leu Leu Ser ThrLeu Asp 1 5 10 15 Pro Gly Gly Lys Thr Gln Asp Glu Leu Asp Lys Gly AlaAla Glu Ala 20 25 30 Glu Leu Asn Lys Lys Val Glu Ala Leu Pro Asn Pro ValXaa Glu Leu 35 40 45 Glu Glu Glu Leu Ser Pro Pro Glu Asp Asn Leu Lys AspAla Glu Thr 50 55 60 Asn His Val Glu Asp Tyr Ile Lys Glu Gly Leu Glu GluAla Ile Ala 65 70 75 80 Thr Lys Gln Ala Glu Leu Glu Glu Thr Pro Gln GluVal Asp Ala Ala 85 90 95 Leu Asn Asp Leu Val Pro Asp Gly Gly Glu Glu Glu100 105 27 119 PRT Streptococcus pneumoniae 27 Leu Glu Asp Ser Gly LeuGly Leu Glu Lys Val Leu Ala Thr Leu Asp 1 5 10 15 Pro Gly Gly Glu ThrPro Asp Gly Leu Asp Lys Glu Ala Ser Glu Asp 20 25 30 Ser Asn Ile Gly AlaLeu Pro Asn Gln Val Ser Asp Leu Glu Asn Gln 35 40 45 Val Ser Glu Leu AspArg Glu Val Thr Arg Leu Pro Ser Asp Leu Lys 50 55 60 Asp Thr Glu Gly AsnAsn Val Gly Asp Tyr Val Lys Gly Gly Leu Glu 65 70 75 80 Lys Ala Leu ThrAsp Glu Lys Val Gly Leu Asn Asn Thr Pro Lys Ala 85 90 95 Leu Asp Thr AlaPro Lys Ala Leu Asp Thr Ala Leu Asn Glu Leu Gly 100 105 110 Pro Asp GlyAsp Glu Glu Glu 115 28 96 PRT Streptococcus pneumoniae 28 Gln Ala LeuTyr Glu Ser Thr Gln Glu Gln Ile Glu Glu Leu Lys Asp 1 5 10 15 Tyr AsnGlu Gln Ile Ser Glu Gly Glu Glu Thr Leu Ile Leu Ala Ile 20 25 30 Gln AsnLys Ile Ser Asp Leu Asp Asp Lys Ile Ala Glu Ala Glu Lys 35 40 45 Lys LeuAla Asp Ser Gln Asn Gly Glu Gly Val Glu Asp Tyr Trp Thr 50 55 60 Ser GlyAsp Glu Asp Lys Leu Glu Lys Leu Gln Ala Glu Gln Asp Glu 65 70 75 80 LeuGln Ala Glu Leu Asp Gln Leu Leu Asp Glu Val Asp Gly Gln Glu 85 90 95

We claim:
 1. A vaccine or immunogenic composition comprising at least afirst isolated immunogenic fragment of PspA and a second isolatedimmunogenic fragment of PspA from S. pneumoniae strains from at leasttwo PspA families.
 2. The vaccine or immunogenic composition of claim 1,wherein the composition comprises an isolated fragment of PspA, whereinthe PspA is from strain Rx1 (ATCC 55834).
 3. The vaccine or immunogeniccomposition of claim 1, wherein the at least two families comprise oneor more clades.
 4. The vaccine or immunogenic composition of claim 3,wherein the composition comprises an isolated fragment of PspA, whereinthe PspA is from strain Rx1 (ATCC 55834).
 5. The vaccine or immunogeniccomposition of claim 3 comprising a minimum of 4 isolated PspA fragmentsfrom said one or more clades.
 6. The vaccine or immunogenic compositionof claim 5, wherein the composition comprises an isolated fragment ofPspA, wherein the PspA is from strain Rx1 (ATCC 55834).
 7. The vaccineor immunogenic composition of claim 3 comprising a minimum of 3 isolatedPspA fragments from said one or more clades.
 8. The vaccine orimmunogenic composition of claim 7, wherein the composition comprises anisolated fragment of PspA, wherein the PspA is from strain Rx1 (ATCC55834).
 9. The vaccine or immunogenic composition of claim 3 comprisinga maximum of 6 isolated PspA fragments from said one or more clades. 10.The vaccine or immunogenic composition of claim 9, wherein thecomposition comprises an isolated fragment of PspA, wherein the PspA isfrom strain Rx1 (ATCC 55834).
 11. The vaccine or immunogenic compositioncomprising at least a first isolated PspA immunogenic fragment and asecond isolated PspA immunogenic fragment from S. pneumoniae strainsfrom at least two PspA families having a C-terminal region of an alphahelix of PspA, wherein the C-terminal region comprises an antigenicepitope of interest.
 12. The vaccine or immunogenic composition of claim11, wherein the composition comprises an isolated fragment of PspA,wherein the PspA is from strain Rx1 (ATCC 55834).
 13. The vaccine orimmunogenic composition of claim 11, wherein the at least two isolatedPspA fragments are immunologically cross-reactive.
 14. The vaccine orimmunogenic composition of claim 11, wherein the at least two familiescomprise one or more clades.
 15. The vaccine or immunogenic compositionof claim 14, wherein the composition comprises an isolated fragment ofPspA, wherein the PspA is from strain Rx1 (ATCC 55834).
 16. The vaccineor immunogenic composition of claim 14, wherein the at least twoisolated PspA fragments are immunologically cross-reactive.
 17. Thevaccine or immunogenic composition of claim 14 comprising a maximum of 6isolated PspA fragments from said one or more clades.
 18. The vaccine orimmunogenic composition of claim 17, wherein the composition comprisesan isolated fragment of PspA, wherein the PspA is from strain Rx1 (ATCC55834).
 19. The vaccine or immunogenic composition of claim 17, whereinthe at least two isolated PspA fragments are immunologicallycross-reactive.
 20. The vaccine or immunogenic composition of claim 14comprising a minimum of 3 isolated PspA fragments from said one or moreclades.
 21. The vaccine or immunogenic composition of claim 20, whereinthe composition comprises an isolated fragment of PspA, wherein the PspAis from strain Rx1 (ATCC 55834).
 22. The vaccine or immunogeniccomposition of claim 20, wherein the at least two isolated PspAfragments are immunologically cross-reactive.
 23. The vaccine orimmunogenic composition of claim 14 comprising a minimum of 4 isolatedPspA fragments from said one or more clades.
 24. The vaccine orimmunogenic composition of claim 23, wherein the composition comprisesan isolated fragment of PspA, wherein the PspA is from strain Rx1 (ATCC55834).
 25. The vaccine or immunogenic composition of claim 23, whereinthe at least two isolated PspA fragments are immunologicallycross-reactive.